You, S., Li, H., Hu, Z. and Zhang, W. (2018). eIF2alpha kinases PERK and GCN2 act on FOXO to potentiate FOXO activity. Genes Cells. PubMed ID: 30043468Summary:PERK and GCN2 are eIF2alpha kinases known to mediate the effects of ER stress and respond to an array of diverse stress stimuli. Previously, it has been reported that ER stress potentiates insulin resistance through PERK-mediated FOXO phosphorylation. Inhibition of PERK improves cellular insulin responsiveness at the level of FOXO activity. This study provides further evidence that FOXO is required for the functional output of PERK by showing that lowering FOXO activity ameliorates a PERK gain-of-function phenotype in Drosophila. More importantly, results are presented demonstrating that GCN2 acts similarly to PERK to promote FOXO activity. Regulation of FOXO by GCN2 is evolutionarily conserved and can be compensated for by PERK. The combination of these mechanisms may contribute to the complex regulatory network between PERK, GCN2, and FOXO, which has been implicated in the development and progression of a variety of diseases.

Progressive decline of proliferative homeostasis in high-turnover tissues is a hallmark of aging, resulting in cancers and degenerative diseases. This is of particular relevance in barrier epithelia, such as the intestinal epithelium, where homeostatic tissue renewal has to be balanced with acute regenerative episodes in response to acute damage or infection. Accordingly, the control of intestinal stem cell (ISC) proliferation has to integrate endogenous control mechanisms with stress and inflammatory signals that promote mitogenic activity of these cells. How cellular stress responses of intestinal epithelial cells (IECs) and intestinal stem cells (ISCs) coordinate and maintain such regenerative processes is a critical question that will provide insight into the etiology of pathologies ranging from inflammatory bowel diseases (IBDs) to colorectal cancers (Wang, 2015).

Long-term homeostasis of the intestinal epithelium is significantly impacted by ER stress. In mouse models for IBDs, ER stress is increased in the intestinal epithelium, and genetic conditions that impair protein folding capacity in the ER of IECs result in complex cell-autonomous and non-autonomous activation of stress signaling pathways, triggering inflammatory conditions similar to IBDs. Recent studies in mice suggest that the UPRER may also influence regenerative processes in the gut directly, as it is engaged in cells transitioning from a stem-like state into the transit amplifying state in the small intestine of mice. In flies, ER stress promotes ISC proliferation, and increased ER stress across the intestinal epithelium is associated with age-related dysplasia in this tissue (Wang, 2014). The downstream signaling mechanisms promoting ISC proliferation in response to ER stress remain unclear (Wang, 2015).

Studies in worms have shown that, in addition to these cell-autonomous responses to ER stress, local activation of the UPRER can trigger UPRER responses in distant tissues, indicating that endocrine processes exist that coordinate such stress responses across cells and tissues. The mechanism(s) regulating and mediating these non-autonomous responses remain elusive (Wang, 2015).

By regulating eIF2α, ATF4 and Nrf2, PERK activation integrates the response to both protein misfolding in the ER and to misfolding-associated oxidative stress. Accumulation of un/misfolded proteins in the ER results in the production of reactive oxygen species (ROS), most likely due to the generation of hydrogen peroxide as a byproduct of protein disulfide bond formation by protein disulfide isomerase (PDI) and ER oxidoreductin 1 (Ero1) (Wang, 2015).

The coordinated control of cellular protein and redox homeostasis by the UPRER and other stress signaling pathways is likely critical to maintain SC function, as the intracellular redox state significantly impacts SC pluripotency, proliferative activity, and differentiation. Recent studies shown that this coordination is achieved in Drosophila ISCs by integration of Nrf2/CncC-mediated responses and Xbp1-mediated ER stress responses (Wang, 2014). The fly orthologue of Nrf2, CncC (Cap 'n' collar isoform-C), counteracts intracellular oxidants and limits proliferative activity of ISCs (Hochmuth, 2011). In ISCs, CncC is inhibited in response to high ER stress (as in Xbp1 loss-of-function conditions), resulting in increased oxidative stress and activation of ISC proliferation (Wang, 2015).

The Drosophila ISC lineage exhibits a high degree of functional and morphological similarities with the ISC lineage in the mammalian small intestine. ISCs self-renew and give rise to transient, non-dividing progenitor cells called EnteroBlasts (EBs) that are lineage-restricted (by Robo/Slit signaling and differential Notch signaling) to differentiate into either absorptive EnteroCytes (ECs) or secretory EnteroEndocrine (EEs) cells. ISCs are the only dividing cells in the posterior midgut of Drosophila and their entry into a highly proliferative state is regulated by multiple stress and mitogenic signaling pathways, including Jun-N-terminal Kinase (JNK), Jak/Stat, Insulin, Wnt, and EGFR signaling (Wang, 2015).

During aging, flies develop epithelial dysplasia in the intestine, caused by excessive ISC proliferation and deficient differentiation of EBs (Biteau, 2008; Choi, 2008). This phenotype is a consequence of an inflammatory condition initiated by immune senescence and dysbiosis of the commensal bacteria, and causes metabolic decline, loss of epithelial barrier function, and increased mortality (Rera, 2012; Biteau, 2010; Guo, 2014), and is associated with a strong tissue-wide increase in ER stress (Wang, 2014). Increasing ER proteostasis in ISCs (by over-expressing Xbp1 or the ERAD-associated factor Hrd1) prevents the age-related over-proliferation of ISCs, suggesting that limiting ER stress-associated signaling in ISCs may be beneficial for tissue homeostasis (Wang, 2015).

This study has tested this hypothesis. The regulation of ISC proliferation by cell-autonomous and non-autonomous UPRER responses was explored in detail, and the consequences of limiting ER stress responses in ISCs for longevity were explored. By analyzing loss of function conditions for Ero1L this study finds that the induction of ISC proliferation by ER stress can be uncoupled from the production of ROS, but that ISC-specific activation of PERK is critical for the proliferative response. Interestingly, PERK activation in ISCs is triggered both by ER stress within ISCs and non-autonomously by ER stress in other cells of the intestinal epithelium, which activate PERK in ISCs through the secretion of Unpaired ligands and activation of JAK/Stat signaling in ISCs. PERK thus integrates epithelial stress responses to control ISC proliferation under challenging proteostatic conditions. Strikingly, PERK is also essential for normal cell proliferation in the ISC lineage, and excessive or chronic PERK activity in ISCs is a cause for the development of epithelial dysplasia in aging flies. Accordingly, this study demonstrates that limiting PERK expression in ISCs is sufficient to extend lifespan (Wang, 2015).

This study identifies the PERK branch of the UPRER as a central node in the control of proliferative homeostasis in the intestinal epithelium, and establishes a previously unrecognized role for PERK in promoting regenerative responses to both tissue-wide and cell-autonomous ER stress. This critical function of PERK in tissue regeneration, however, also results in the aging-associated loss of proliferative homeostasis in the intestinal epithelium, limiting organismal lifespan. The unique and specific increase in eIF2α phosphorylation in ISCs in stressed and aging conditions suggests a differential activation of the PERK-eIF2α branch of the UPRER between ISCs and their daughter cells. It remains unclear whether this differential regulation reflects different strategies in combating ER stress between these cell populations, and additional studies are necessary to address this interesting question (Wang, 2015).

Drosophila ISCs, as many other stem cell types, are controlled extensively by redox signals. Previous work, as well as the results shown in this study, suggests that ER-induced oxidative stress plays a central role in the control of ISC proliferation after a proteostatic challenge. The results support the notion that ER-induced ROS is a consequence of the PDI/Ero1L system, as has been proposed in mammalian cells (Harding, 2003). However, Ero1L, as a thiol oxidase, may also affect the proper folding and maturation of Notch directly (as described previously), inhibiting ISC differentiation, and resulting in stem cell tumors. The phenotype of Ero1L-deficient ISC lineages supports a role for Ero1L in Notch signaling (tumors with elevated numbers of Dl+ cells). At the same time, this study's results also support a role for Ero1L in limiting ISC proliferation directly through the UPRER (and independently of Notch signaling or oxidative signals), as loss of Ero1L induces PERK activity without promoting ROS production in these cells. PERK itself is required for the induction of cell cycle and DNA replication genes in ISCs responding to TM treatment, yet it also induces antioxidant genes under these conditions, suggesting complex crosstalk between PERK-mediated control of mitotic activity of ISCs and the control of redox homeostasis in these cells (Wang, 2015).

The fact that loss of Ero1L activates PERK while not inducing Xbp1 in ISCs suggests selective activation mechanisms for these two branches of the UPRER. The study proposes that this selectivity is associated with the production of ROS and that ER protein stress activates the Xbp1 branch when associated with a ROS signal, while PERK can be activated by unfolded proteins independently of ROS production. Further studies are needed to dissect the relative contribution of ROS production, PERK activation and Notch perturbation in the control of ISC proliferation in Ero1L loss of function conditions (Wang, 2015).

This study highlights the interaction between cell-autonomous and non-autonomous events in the ER stress response of ISCs and support the notion that improving proteostasis by boosting ER folding capacity in stem cells improves long-term tissue homeostasis and can impact lifespan. The regulation of PERK activity in ISCs by the JAK/Stat signaling pathway provides a tentative mechanism for the interaction between IECs experiencing ER stress and ISCs: the study proposes that JNK-mediated release of JAK/Stat ligands from stressed IECs results in JAK/Stat mediated activation of PERK in ISCs, and that this activation is required for the proliferative response of ISCs to epithelial dysfunction. The activation of JAK/Stat signaling in the intestinal epithelium of animals in which Xbp1 is knocked down in ECs, the requirement for JNK activation and Upd expression in ECs for ISC proliferation in response to stress, and the requirement for Stat (and Hop and Dome) in ISCs for the activation of eIF2α phosphorylation and stress-induced ISC proliferation, support this model. The mechanisms by which Stat mediates activation of PERK remain unclear, and will be interesting topics of further study (Wang, 2015).

Studies in worms have established the UPRER as a critical determinant of longevity, and Xbp1 extends lifespan by improving ER stress resistance. This study's data further support the notion that regulating ER stress response pathways is critical to increase health- and lifespan. Here, chronic PERK activation can be considered a downstream readout of the buildup of proteotoxic stress in the intestinal epithelium during aging, which then perturbs proliferative homeostasis by continuously providing pro-mitotic signals to ISCs. Knocking down PERK in ISCs limits these pro-mitotic signals, improving homeostasis and barrier function, and extending lifespan. Lifespan is generally extended when ISC proliferation is limited in older flies, but not when it is completely inhibited. Accordingly, they observe lifespan extension when PERK is knocked down using an RNAi approach that does not completely ablate PERK function.

ER stress has been documented as tightly associated with intestinal inflammation and the development of IBDs in mice and humans. Genetic variants in Xbp1 are associated with higher susceptibility to IBD and a recent study indicates that Xbp1 can act as a tumor suppressor in the intestinal epithelium, by limiting intestinal proliferative responses and tumor development through the control of local inflammation. In this context, the specific role of PERK in the control of ISC proliferation in the fly gut is consistent with the function of PERK in the intestinal epithelium of mice, where activation of PERK can promote transition of ISCs into the transient amplifying cell population. While the Drosophila midgut epithelium does not contain a transit amplifying cell population, this study's data suggest that a role for PERK in the proliferative response of the ISC lineage to ER stress is conserved (Wang, 2015).

Due to the importance of the UPRER in the maintenance of tissue homeostasis in aging organisms, therapies targeting the UPRER are promising strategies to delay the aging process. Accordingly, pharmaceuticals that can limit ER stress (such as Tauroursodeoxycholic acid, TUDCA and 4-phenylbutyrate, PBA) have had therapeutic success in various human disorders. Interestingly, flies fed PBA show increased lifespan, yet the effects of PBA on intestinal homeostasis have not yet been explored. Based on this work, it is likely that further characterization of the effects of UPRER-targeting drugs on ISC function and intestinal homeostasis will help develop clinically relevant strategies to limit human aging and extend healthspan (Wang, 2015).

The endoplasmic reticulum (ER) has a major role in protein folding. The accumulation of unfolded proteins in the ER induces a stress, which can be resolved by the unfolded protein response (UPR). Chronicity of ER stress leads to UPR-induced apoptosis and in turn to an unbalance of tissue homeostasis. Although ER stress-dependent apoptosis is observed in a great number of devastating human diseases, how cells activate apoptosis and promote tissue homeostasis after chronic ER stress remains poorly understood. This study used the Drosophila wing imaginal disc as a model system. Presenilin overexpression induces chronic ER stress in vivo. In this novel model of chronic ER-stress, a PERK/ATF4-dependent apoptosis required downregulation of the antiapoptotic diap1 gene. PERK/ATF4 also activated the JNK pathway through Rac1 and Slpr activation in apoptotic cells, leading to the expression of Dilp8. This insulin-like peptide caused a developmental delay, which partially allowed the replacement of apoptotic cells. Thanks to a novel chronic ER stress model, these results establish a new pathway that both participates in tissue homeostasis and triggers apoptosis through an original regulation (Demay, 2014).

As has been reported in mammalian cells, this study hase validated that Psn overexpression can provoke chronic ER stress in Drosophila. In mammalian models, the UPR branches can display opposite roles depending on the model. For example, Perk can be either anti or proapoptotic (Hamanaka, 2009; Verfaillie, 2013; Oomen, 2013) Thanks to a new model of chronic ER stress, this study has demonstrated that the PERK/ATF4 pathway has a fundamental role in Drosophila tissue homeostasis. So far, the autosomal dominant retinitis pigmentosa (ADRP) model was the only model of strong chronic ER stress reported in Drosophila. This study has validated that Psn overexpression can also provoke a chronic ER stress in Drosophila, as previously reported in mammalian cells. In both Drosophila models, apoptosis is induced by UPR in response to ER stress. Nevertheless, this induction involves totally different pathways. In this study's chronic ER stress model, cell death induction is PERK/ATF4 dependent and JNK independent, contrarily to the ADRP model in which CDK5 activates JNK signaling that triggers apoptosis. These differences show that the complexity of ER stress-induced signaling found in mammals is conserved in Drosophila, thus highlighting the usefulness of ER stress models plurality (Demay, 2014).

This study has shown that the PERK/ATF4 pathway induces a caspase-dependent apoptosis by repressing diap1 transcription. However, PERK has been described to exert some antiapoptotic activity by inducing IAP gene expression in mammals. This effect does not seem to rely on direct targets of PERK, ATF4 and CHOP. Similarly, this study did not find any ATF4 consensus binding sequence (5'-RTTRCRTCA-3') in the diap1 promoter region, and no CHOP homolog has been found in Drosophila. Therefore, the mechanisms involved in PERK regulation of IAPs remain to be clarified (Demay, 2014).

In the chronic ER stress model, the JNK pathway is activated in apoptotic cells to favor tissue homeostasis without stimulating cell proliferation. This is in contrast to a JNK activation in cells neighboring apoptotic cells, which results in an increase of the proliferation rate. Similar to this observation, JNK activation in apoptotic cells has been observed in 'undead cell' models. In these models, the JNK pathway could be activated by DIAP1 or DRONC, whereas JNK signaling seems to be primarily independent from DIAP1/DRONC in the current model. In a mammalian model of chronic ER stress, the IRE1 branch of the UPR activated the JNK pathway to trigger apoptosis thanks to TRAF2/ASK1. In the ER stress model, depletion of traf2 or ask1 had no effect. Instead, this study has shown that JNK pathway activation mainly depends on the PERK/ATF4 pathway. Interestingly, this particular JNK pathway is not mainly activated by apoptosis and does not modulate cell death or proliferation (Demay, 2014).

It was also shown that PERK/ATF4 regulates an ER stress-induced developmental delay. As previously reported, it was observed that Dilp8 is a major contributor to developmental delay. An obvious candidate for a Dilp8-independent developmental delay regulation was the retinoic acid signaling that has been reported to modulate an irradiation-induced developmental delay. This study tested if this pathway could also regulate the developmental delay caused by Psn overexpression. No wing phenotype modification was detected upon the downregulation of this pathway (Demay, 2014).

This study characterized the components of the JNK signaling that is activated in response to chronic ER stress in Drosophila wing imaginal discs (see Model of tissue homeostasis maintenance after an ER stress). The small GTPase Rac1 would activate the JNKKK Slpr, which in turn would activate JNK signaling core to regulate dilp8 expression and ultimately favor development delay and tissue homeostasis maintenance. How the ATF4/PERK branch activates Rac1 remains to be elucidated. The results also suggest the existence of a negative feedback loop regulating the JNK pathway, which would involve the JNKKKK, Msn. This is in agreement with a genetic and phosphoproteomic study showing that Msn is able to inhibit the phosphorylation of Jun. Considering that the JNK pathway induces dilp8 expression in abnormally growing imaginal discs in other stress models, one may wonder whether the same JNK pathway is implicated in these models. Moreover, one may wonder whether dilp8 control during tissue homeostasis-associated developmental delay is always JNK-dependent and relies on a Rac1/Slpr pathway (Demay, 2014).

To summarize, this study has shown that in response to an ER stress induced by Psn overexpression, the PERK pathway is activated resulting in a Janus-faced ATF4 role. On one hand, ATF4 induces caspase-dependent apoptosis by repressing diap1 expression and on the other hand, it favors tissue homeostasis maintenance through the induction of a Rac1/Slpr/JNK pathway and the resulting dilp8 expression. More investigations on this new Drosophila chronic ER-stress model should allow the identification of novel regulators of UPR-dependent tissue and organism homeostasis that may be conserved in mammals (Demay, 2014).

The Hedgehog signalling pathway regulates autophagy

Autophagy is a highly conserved degradative process that removes damaged or unnecessary proteins and organelles, and recycles cytoplasmic contents during starvation. Autophagy is essential in physiological processes such as embryonic development but how autophagy is regulated by canonical developmental pathways is unclear. This study shows that the Hedgehog signalling pathway inhibits autophagosome synthesis, both in basal and in autophagy-induced conditions. This mechanism is conserved in mammalian cells and in Drosophila, and requires the orthologous transcription factors Gli2 and Ci, respectively. Furthermore, it was demonstrated activation of the Hedgehog pathway reduces PERK levels, concomitant with a decrease in phosphorylation of the translation initiation factor eukaryotic initiation factor 2alpha, suggesting a novel target of this pathway and providing a possible link between Hedgehog signalling and autophagy (Jimenez-Sanchez, 2012).

In Drosophila, a single Ptch receptor responds to Hh molecules, whereas in mammalian cells Ptch1 and Ptch2 share this function. Although knockdown of Ptch1 did not inhibit autophagy in basal conditions, activation of the Hh pathway was not as efficient with Ptch1 compared with Ptch2 knockdown. Also, Ptch1 is a transcriptional target of the Hh pathway and its expression is expected to increase when the pathway is activated by Ptch1 siRNA treatment. This feedback implies that Ptch1 siRNA may not be efficient enough to completely counteract its own transcriptional activation. Although more detailed experiments would be needed to rule out a differential contribution of these receptors, the data suggest that Shh modulates autophagy through Ptch1 and/or Ptch2 (Jimenez-Sanchez, 2012).

Gli transcription factors have distinct activator and repressor functions and their roles differ during embryonic development. Although a contribution of Gli1 and Gli3 to autophagy regulation cannot be completely excluded, it was confirmed that Gli2 is necessary for the inhibition of autophagy by Hh signalling. The increased LC3-II levels in Gli2-knockout embryos further confirmed the data in a biologically relevant context. These data do not explain the developmental defects in these mice but might suggest that some of their phenotypes could be secondary to autophagy dysregulation. However, almost certainly, factors other than autophagy inhibition will have major contributions to the developmental defect in these Gli2-knockout mice. Equally, it cannot be excluded that other pathways that increase autophagy could be aberrantly activated in these embryos (Jimenez-Sanchez, 2012).

Despite the effects of Gli2 and Ci on autophagy, the role of their upstream activator Smo was less clear-cut. This is not surprising, as it has been shown that Smo levels are not sufficient to stimulate the pathway, but an activation process is required, with Ptch influencing the transition to an active state of Smo in response to Shh. In Drosophila, null mutations in Smo did not influence autophagy whereas Costal2 inhibition did, and it is therefore also possible that the Hh/Ptch complex and Ci may partially bypass Smo in Drosophila, consistent with the effects of this pathway on Costal2 localization to endomembranes. Consequently, the data suggest that canonical and non-canonical Hh mechanisms might be involved in regulating autophagy, or that other factors might be necessary in Hh-mediated regulation of autophagy (Jimenez-Sanchez, 2012).

Hh inhibition has been largely pursued as a therapeutic strategy for types of cancer resulting from a hyperactivation of the pathway, such as basal cell carcinomas or medulloblastoma. Although the roles of autophagy in cancer are controversial and complex, it is interesting to consider whether inhibition of autophagy by Hh would exacerbate or prevent the progression of these tumours when searching for pharmacological strategies. Cyclopamine, which binds and inhibits Smo, is one of the best characterized Hh signalling inhibitors and it has been shown to be protective in Hh-related cancers. As expected, cyclopamine caused LC3-II
(microtubule-associated protein 1 light chain 3 lipidated form, a product of autophagy). In the presence of bafA1, the increase was not as large as in non-treated cells. This result was unexpected because bafA1 should exacerbate the changes on LC3-II if cyclopamine is triggering autophagosome synthesis, suggesting that cyclopamine might also affect autophagosome maturation. Consistent with an impairment in autophagosome degradation, cyclopamine and bafA1 both similarly reduced the number of acidic vesicles in mRFP-GFP-LC3 cells. These data suggest that cyclopamine affects autophagosome degradation but that this effect is independent of Hh inhibition, as cyclopamine could increase LC3-II levels even in Gli2-null MEFs. These data suggest that cyclopamine has two opposite effects on autophagy. It increases autophagosome synthesis by inhibiting Smo activity, but it simultaneously impairs autophagosome maturation through an unknown mechanism that is independent of Gli2. Effects of cyclopamine independent of Smo inhibition have been reported in human breast cancer cell lines and in zebrafish, suggesting that this drug has additional molecular targets (Jimenez-Sanchez, 2012).

PRK-like ER kinase (PERK or EIF2AK3) is an ER-resident protein responsible for the phosphorylation of eukaryotic initiation factor 2α (eIF2α), which inhibits protein translation in response to ER stress, In search of potential Hh transcriptional targets that could modulate autophagy, PERK was identified as a consistent potential target and, in agreement with previous observation, PERK is repressed upon Gli1 and Gli2 induction. It has also been suggested that expression of Gli2 repressed a considerably larger number of genes than Gli1, an observation that may be of relevance when considering the more dominant effect of Gli2 on autophagy. Although Gli2 acts mainly as a transcriptional activator upon Hh activation, it can be proteolytically processed into a transcriptional repressor, similar to the dual function of Ci in Drosophila. The autophagy inhibitory action of Gli2 can be explained by either a gain of repressor function towards some genes (such as PERK) in its active form, or by the fact that when activated it leads to increased expression of a second transcriptional repressor. It remains to be elucidated which of these mechanisms is in action in the case of PERK transcription (Jimenez-Sanchez, 2012).

In response to unfolded proteins in the ER lumen, PERK is activated and phosphorylates eIF2α, increasing translation of the transcription factor ATF4. The exact mechanism by which eIF22α controls autophagy is unknown, eIF22α regulates Atg12 levels in the presence of expanded polyglutamines and ATF4 has been suggested to increase transcription of Atg5 and LC3. Indeed PCR Array data show that levels of >Atg5 upon Shh ligand are decreased to 0.8-fold compared with control cells. However, it is unlikely that this is the complete mechanism, as it has yet to be conclusively demonstrated that transcriptional induction of LC3 and Atg5 upregulate autophagy (Jimenez-Sanchez, 2012).

It is also possible that PERK interacts more directly with other signalling pathways involved in autophagy regulation. As an example, the transcription factor Nrf2, known to be involved in autophagy regulation, is phosphorylated by PERK independently of eIF22α in response to ER stress, promoting its import to the nucleus and activation of transcription (Jimenez-Sanchez, 2012).

In conclusion, these data not only provide insights into the connection between Hh and autophagy but also into the potential implications that Hh has in controlling protein homeostasis in physiological and disease conditions (Jimenez-Sanchez, 2012).

Cell apoptosis, or programmed cell death (PCD), is one of the most fundamental processes essential for normal development in multicellular organisms. Cell apoptosis is tightly controlled to eliminate excess or damaged cells during development, while dysregulation can lead to pathological diseases, such as neurodegenerative diseases and cancer. Although regulation of cell apoptosis involves a sophisticated and complex molecular cascade, the core machinery that initiates, mediates and executes programmed cell death as well as related regulatory pathways are evolutionary conserved among species. Drosophila melanogaster, with its well-annotated genome and established genetic tools, is commonly used to study mechanism of cell apoptosis and discover novel regulatory pathways and genes for regulating development and tissue homeostasis (Zhang, 2016).

The defender against apoptotic cell death 1 (Dad1) gene was first identified and considered as a negative regulator of cell apoptosis from a temperature-sensitive hamster cell line, tsBN7. With the temperature sensitive mutation in Dad1 gene, cells are normal at permissive temperature, while shifting to restrictive temperature led to programmed cell death. Homologous proteins of hamster Dad1 from other species were found to be highly conserved in sequence and function. The yeast homologue of Dad1 is called Ost2 with 40% identity compared to hamster Dad1 and ost2 mutant induces yeast cell apoptosis. The function of Dad1 has been studied in multicellular organisms as well. Mouse Dad1 mutants exhibited developmental delay, aberrant morphology and increased cell apoptosis during embryogenesis and only survived up to stage of midgestation. In Caenorhabditis elegans, overexpression of either Caenorhabditis Dad1 or human Dad1 induced by a heat-shock promoter inhibited developmental cell death during embryogenesis. While ectopic expression of Dad1 in mouse thymocytes did not increase survival of T cells under apoptotic stimuli, peripheral T cells from spleen and lymph nodes in Dad1 transgenic mice increased cell proliferation. These results indicate that Dad1 plays a critical role in regulating cell viability and apoptosis (Zhang, 2016).

Dad1 is identified as a subunit of oligosaccharyltransferase complex (OST), which acts at the first step for protein N-linked glycosylation. N-linked glycosylation is an important protein modification process, which transfers oligosaccharide to selected asparagine of target nascent proteins to ensure their proper folding and maturation in the endoplasmic reticulum (ER). In eukaryotes, OST complex has a catalytic subunit Stt3 as a core and other six non-catalytic subunits, including Ribophorin I, Ribophorin II, Ost48, Dad1/Ost2, N33/Ost3, and Ost4. There are two OST catalytic subunit genes in vertebrates and insects, referred to as Stt3A and Stt3B. Non-catalytic subunits facilitate the catalytic function of Stt3A and Stt3B. Among them, Ost48 and Dad1 are critical for assembly and stability of Stt3 as well as the OST complex, and loss of them can lead to hypoglycosylation. Ribophorin I escorts the N-glycosylation of OST complex on specific substrates, especially some membrane proteins. Because loss of any one non-catalytic subunit does not fully abolish the function of OST, it is possible that they may have redundant roles or different non-catalytic subunits assist the glycosylation of specific targets (Zhang, 2016).

Dad1, as an N-glycosylation regulatory protein, plays an important role in cell survival, but up to now no detailed mechanism is known about how loss of Dad1 induces cell apoptosis. Based on the evidence from studies in different organisms, Dad1 may facilitate the OST complex to target specific proteins that directly maintain cell survival. Another possibility is that accumulation of unfolded or misfolded proteins of hypoglycosylation at ER triggers stress signaling and initiates programmed cell death. It is also possible that Dad1 affects cell viability in an OST-independent manner. For instance, Dad1 interacts with a Bcl2 family protein, Mcl1, which might lead to apoptosis inhibition (Zhang, 2016).

This study used Drosophila Dad1 (dDad1), which shows more than 70% identity with human DAD1, to study how loss of Dad1 function affects cell viability and tissue growth. Loss of Dad1 in Drosophila was found to decreased tissue growth due to induction of apoptosis and absence of cell proliferation. dDad1 is required for efficient N-glycosylation in developing tissues and the small wing phenotype induced by dDad1 knock-down can be enhanced by reducing gene dosage of the OST catalytic subunit. In addition, the c-Jun N-terminal kinase (JNK) pathway was found to mediate dDad1 knockdown-induced cell apoptosis in wing discs through mitogen-activated protein kinase kinase kinase 1 (Mekk1) and wallenda (wnd). Moreover, in dDad1 knock-down tissues, ER stress is induced and Perk-Atf4 pathway functions upstream of the JNK pathway. Intriguingly, unfolded protein response (UPR) signaling appears to play a dual role in inducing cell death and stimulating compensatory proliferation of neighboring cells for tissue homeostasis (Zhang, 2016).

This study found that loss of dDad1 in the wing interferes with N-linked protein glycosylation, which triggers ER stress and activates the downstream JNK pathway to cause apoptosis. Interestingly, although this study suggests that dDad1 regulates tissue growth through the OST complex, only one of two catalytic subunits, Stt3A (CG1518), genetically interacts with dDad1 in growth control. With the association of dDad1, Stt3A may become the major functioning complex modifying crucial proteins or more nascent proteins in developing tissues, therefore inducing severe or prolonged ER stress if dDad1 activity is absent. Different tissues or regions of tissues may have different levels of sensitivity to the loss of dDad1, which could explain why cell apoptosis was more frequently observed in wing pouch than other region of the wing disc in these experiments. Similar phenomena were also reported in other studies. For instance, Dad1 mutant mice exhibit cell death which leads to developmental defect and embryonic lethality, but the death of cells only happens in some tissues of the embryo (Zhang, 2016).

One study reports that dDad1 homozygous mutant clones in larval fact body induced cell autophagy. However, the current study did not observe cell autophagy using lyso-tracker staining in dDad1 mutant clones in larval wing discs. The different response may be due to different tissue-specific context. Therefore, apoptosis induced by loss of dDad1 in the wing is unlikely to be dependent of autophagy process (Zhang, 2016).

As an N-glycosylation regulator, dDad1 may have other target proteins essential for animal viability. In this study, although blocking Perk-Atf4 or JNK pathway suppresses apoptosis induced by loss of dDad1, the lethality of en-Gal4/UAS-dDad1 RNAi individuals cannot be rescued. Thus, cell apoptosis induced by loss of dDad1 may not be the major reason for causing lethality of the mutant organism. It would be interesting to identify potential targets of dDad1 that are essential for normal development of Drosophila (Zhang, 2016).

The Perk-Atf4 pathway may play both protective and destructive roles in growth control in loss of dDad1 model: on one hand, it activates JNK signaling to cause cell apoptosis; on the other, it may send out signals to neighboring cells to increase cell proliferation (Zhang, 2016).

In Drosophila, it is known that prolonged ER stress induces cell apoptosis, however the mechanism of ER stress activates apoptosis remains elusive. A few Drosophila ER stress models have been established and their connection with apoptosis has been validated. Nevertheless, they suggested different pathways leading to cell death and none of them is the same as what was found in the current study. In a chronic ER stress model due to Presenilin (Psn) overexpression, cell apoptosis is induced in a Perk-Atf4 dependent but JNK-independent manner. Although the study found that the JNK pathway is activated by Perk-Atf4, it induces dilp8 expression which extends developmental stage to keep tissue homeostasis, rather than stimulates cell death. In the current study, induction of cell apoptosis in the absence of dDad1 function was found to be dependent on both Perk-Atf4 and JNK pathways. The JNK pathway acts downstream of Perk-Atf4 and causes cell apoptosis. It was also found that Mekk1 and Wnd are two major JNKKK's that activate JNK pathway in response to loss-of-dDad1 induced ER stress. This is consistent with the observations from another ER stress model, autosomal dominant retinitis pigmentosa (ADRP) model. However, ADRP model suggests that activation of JNK pathway and cell apoptosis does not rely on UPR signaling. In mammalian models of ER stress, cell apoptosis is induced through activation of IRE1-XBP1 branch, followed by JNK pathway activation via TRAF2/ASK1. In the current study, it was not possible to test the role of Ire1-Xbp1 branch due to the embryonic lethality caused by expression of Ire1 RNAi or Xbp1 RNAi driven by en-Gal4, although its activation was observed. Ask1 does not seem to play a role in mediating cell apoptosis in the loss-of-dDad1 model, since knocking down of Ask1 cannot restore loss-of-dDad1 induced wing size deduction (Zhang, 2016).

Compensatory proliferation is a self-rescue mechanism to keep tissue homeostasis when some cells are under stress or apoptosis. Mechanism of compensatory proliferation was mostly studied in Drosophila. In fly wing discs, Dronc in the apoptotic cells stimulates JNK pathway, inducing expression of mitogens, Dpp and Wg, to presumably promote proliferation of neighboring cells. Interestingly, in this study, activation of Perk-Atf4 pathway appears to play a role in inducing extra cell proliferation but does not rely on activation of the JNK pathway nor cell death. It suggests that Atf4 transcription factor may induce transcription of mitogenic genes to stimulate cell proliferation, in addition to the genes that are related to stress response (Zhang, 2016).

N-glycosylation of Wg was affected in dDad1 mutant cells. However, it turned out that N-glycosylation of Wg is unimportant for its secretion and signaling in fly tissues. Regardless, it is unlikely that Wg was responsible for mediating extra cell proliferation in this loss-of-dDad1 model based on the following observations. Firstly, compensatory cell proliferation can still occur in the absence of wg and dpp in tissues undergoing massive cell apoptosis. Moreover, this study found that proliferation of extra cells can still occur when JNK signaling was blocked, while the JNK pathway is presumably responsible for Wg induction. Therefore, further studies are needed to address how Perk-Atf4 signaling acts to induce compensatory cell proliferation to maintain tissue homeostasis (Zhang, 2016).

Some studies suggest that Dad1 may have a direct effect on cell death suppression. Overexpression of hDAD1 or ceDAD1 has been shown to be sufficient to inhibit developmental cell apoptosis in C. elegans. Overexpression of DAD1 in mouse thymocyte did not increase survival of T cells under apoptosis stimuli, however peripheral T cells from spleen and lymph node in Dad1 transgenic mice have increased cell proliferation. This work examined the phenotypes of dDad1 transgenic flies in different tissues. Drosophila pupa eye is a good model to study developmental apoptosis because wild-type flies have fixed number of interommatidial cells in mid-pupal eye discs due to the elimination of excess cells by apoptosis. This study found that overexpression of dDad1 in the eye did not cause the accumulation of extra interommatidial cells in mid-pupal eye discs, which indicates that dDad1 is not sufficient to block normal apoptosis during eye development. In addition, this study overexpressed apoptosis-inducing genes in Drosophila wing and eye to test if dDad1 is able to suppress induced apoptosis. The cell apoptosis in the wing induced by overexpression of UAS-eiger failed to be suppressed by dDad1 overexpression. However, overexpression of dDad1 could moderately rescue cell death induced by GMR-hid in adult eyes. These observations indicate that Drosophila dDad1 inhibits induced cell apoptosis moderately and conditionally, but exhibits no effect on normal developmental apoptosis. Therefore, these observations suggest that dDad1 may not function as an active defender of cell apoptosis in Drosophila (Zhang, 2016).

Metazoans respond to various forms of environmental stress by inducing the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) at serine-51, a modification that leads to global inhibition of mRNA translation. This study demonstrates induction of the phosphorylation of eIF2alpha in mammalian cells after either pharmacological inhibition of the phosphoinositide 3-kinase (PI3K)-Akt pathway or genetic or small interfering RNA-mediated ablation of Akt. This increase in the extent of eIF2alpha phosphorylation also occurred in Drosophila cells and depends on the endoplasmic reticulum (ER)-resident protein kinase PERK, which is inhibited by Akt-dependent phosphorylation at threonine-799. The activity of PERK and the abundance of phosphorylated eIF2alpha (eIF2alphaP) were reduced in mouse mammary gland tumors that contained activated Akt, as well as in cells exposed to ER stress or oxidative stress. In unstressed cells, the PERK-eIF2alphaP pathway mediates survival and facilitates adaptation to the deleterious effects of the inactivation of PI3K or Akt. Inactivation of the PERK-eIF2alphaP pathway increases the susceptibility of tumor cells to death by pharmacological inhibitors of PI3K or Akt. Thus, it is suggested that the PERK-eIF2alphaP pathway provides a link between Akt signaling and translational control, which has implications for tumor formation and treatment (Mounir, 2011).

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. This study reports the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA (N-methyl-D-aspartate receptor-associated protein) synergies with TMBIM6/BI-1 (Bax Inhibitor-1) in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane (Rojas-Rivera, 2012).

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. This study has developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2alpha phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest (Malzer, 2010).

Four distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases phosphorylate eIF2alpha at S51 and regulate protein synthesis in response to various environmental stresses. These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticulum (ER)-resident kinase (PERK) and the GCN2 protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes. This study has characterized the second eIF2alpha kinase found in Drosophila, a PERK homologue (DPERK). Expression of DPERK is developmentally regulated. During embryogenesis, DPERK expression becomes concentrated in the endodermal cells of the gut and in the germ line precursor cells. Recombinant wild-type DPERK, but not the inactive DPERK-K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2alpha at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2. The full length protein, when expressed in 293T cells, located in the ER-enriched fraction, and its subcellular localization changed with deletion of different N-terminal fragments. Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function. Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress. Furthermore, the stable complexes between wild-type DPERK and DPERK-K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2alpha kinase activity (Pomar, 2003).

In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). Recently, a new family member, pancreatic eIF-2alpha kinase (PEK) has been identified from rat pancreas. PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER. This study identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans. Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues. In mammalian cells subjected to ER stress, elevated eIF-2alpha phosphorylation was found to be coincident with increased PEK autophosphorylation and eIF-2alpha kinase activity. Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen. To address the role of C. elegans PEK in translational control, this kinase was expressed in yeast, and it was found to inhibit growth by hyperphosphorylation of eIF-2alpha and inhibition of eIF-2B. Furthermore, vaccinia virus K3L protein, an inhibitor of the eIF-2alpha kinase PKR involved in an anti-viral defence pathway, was also found to reduce PEK activity. These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway (Sood, 2000).